31 The importance of testing cryoprotectants when developing a sperm-freezing protocol for squamates

Nearly 10 000 reptilian species in the order Squamata have been assessed by International Union for Conservation of Nature (IUCN), revealing that 22% are threatened with extinction and another 14.9% are data deficient. There is an increasing need to establish a sperm-freezing protocol for snakes and lizards to preserve their genetic diversity. Three species in the Viperidae family (Ethiopian mountain adder, Mangshan pit viper, and southern pacific rattlesnake) and two species in the Varanidae family (white-throated monitor and black tree monitor) were included in this study to evaluate the effect of cryoprotectants. Sperm was opportunistically collected postmortem and immediately assessed for motility score (MS, motility × speed of progression²), plasma membrane integrity (PL), and acrosome integrity (AC) before freezing. Sperm from each male was extended in a TEST yolk buffer in 500 µL with a final percentage of 12% glycerol (GLY), 12% dimethyl sulfoxide (DMSO), or 6% DMSO:6% GLY and frozen in cryovials at 0.3°C min⁻¹ to −40°C before liquid nitrogen storage. For each treatment, triplicate vials were thawed at 37°C for 90 s, cryoprotectant was removed by centrifugation (60 g), and the sperm pellet was resuspended in M199+ HEPES. Sperm was evaluated at 22°C (Viperidae) or 37°C (Varanidae) immediately following resuspension (T0) and at 60 min (T60). All data were expressed as a percentage of initial (%IMS, %IPL, and %IAC for MS, PL, and AC, respectively). The effect of treatment on %IMS, %IPL, and %IAC was analysed by ANOVA and Tukey’s HSD test. For the Viperidae family, treatment significantly affected %IMS at T0 (P = 0.0009) only, with 12% GLY maintaining greater motility scores than 12% DMSO. The value %IPL was significantly affected by treatment at T0 (P = 0.0005) and T60 (P = 0.0008), with 12% GLY retaining greater plasma membrane integrity than the other two treatments at both time points. Acrosome integrity was not significantly affected by treatment (P = 0.2429). In the Varanidae family, treatment significantly affected %IMS at T0 (P < 0.0001) and T60 (P < 0.0001), with 12% DMSO and 6% DMSO:6% GLY maintaining greater motility scores than 12% GLY at T0 and T60. Treatment did not affect %IPL at T0 (P = 0.3296) but did at T60 (P = 0.0014) where 12% DMSO and 6% DMSO:6% GLY maintained greater plasma membrane integrity than 12% GLY. Treatment significantly affected %IAC (P < 0.0001) with 12% DMSO preserving a significantly greater proportion of intact acrosome than the other cryoprotectant treatments. These preliminary results suggest that GLY is the preferred cryoprotectant for Viperidae sperm (% of initial motility, 55% GLY, 17% DMSO) while DMSO supports greater cryosurvival of Varanidae sperm (% of initial motility, 69% DMSO, 22% GLY). This study highlights the significance of testing different cryoprotectants while developing a sperm-freezing protocol for squamates, as there are distinct differences between taxonomic families.